Filoviruses for which there is no cure or treatment cause fatal hemorrhagic fevers in primates. The life cycle of these viruses is a complex process requiring production of infectious virions from the host cell membrane to sustain the infection. The process of generating a new virion involves an array of protein interactions but there is a paucity of mechanistic information on this process. Because inhibition of new virion formation from the plasma membrane of the host cell is an avenue of curing and preventing these infections, identifying the key interactions and modes of inhibition are of utmost importance. For an infectious virion to form, newly synthesized viral proteins and genomic RNA in the form of nucleocapsids are transported to the cell membrane to form a bud site. Generation of the bud site is a prime target for therapy and has been speculated to require any array of membrane-protein interactions. We demonstrate two proteins from Ebola termed matrix proteins;VP40 and VP24 associate with specific lipid membranes and induce changes in membrane curvature to generate virus like particles (VLPs) (a model of new virions). This discovery and subsequent investigation will create a new paradigm in the life cycle of a virus. Moreover, from studying the viral matrix proteins much new information can be obtained on mechanisms of peripheral protein assembly impacting more than one scientific community. The primary objective of this proposed research is to fully elucidate the mechanistic basis of lipid-interactions by filoviridae matrix proteins and their mechanism of generating VLPs from host cells.
The specific aims for the proposed research are as follows: 1) Determination of the membrane targeting and curvature inducing mechanism of the matrix protein VP40;2) Determination of the interplay of cellular membranes and F-actin in targeting VP24 and its ability to curve membranes;3) Determination of the lipid-dependent interaction properties of VP40 and human Nedd4-1 in the generation of VLPs. The principal methodologies to be used include: (1) the biophysical analysis of interactions of viral proteins (VP40 and VP24) with various model membranes by monolayer, surface plasmon resonance, sedimentation, and stop-flow analysis;(2) the cellular membrane targeting properties of VP40 and VP24 using fluorescent proteins;(3) the qualitative and quantitative membrane curvature inducing properties of these proteins in vitro and in mammalian cells;(4) the structural investigation of the membrane curvature process with X-ray and an array of fluorescence methodologies;(5) the hijacking of the human protein Nedd4-1 by VP40 on lipid membranes both in vitro and in mammalian cells;(6) the interaction properties of VP24 with F-actin. Collectively, these aims will provide a comprehensive view of the plasma membrane based viral assembly and generation of new virions.
There is a paucity of therapeutics for treatment or prevention of many viral infections and there is increasing concern countries or terrorist groups may have weaponized viruses for dissemination. The proposed studies will provide the first mechanistic insight into how viral proteins interact with mammalian cell membranes to generate new infectious virions serving as a prerequisite to the development of therapeutic protocols for generation of small-molecule inhibitors, vaccines, or therapeutic antibodies.
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